www.nature.com/scientificreports OPEN Multi‑pronged approach to human mesenchymal stromal cells senescence quantifcation with a focus on label‑free methods Weichao Zhai1, Jerome Tan1, Tobias Russell2, Sixun Chen1, Dennis McGonagle2, May Win Naing1,3, Derrick Yong3* & Elena Jones2* Human mesenchymal stromal cells (hMSCs) have demonstrated, in various preclinical settings, consistent ability in promoting tissue healing and improving outcomes in animal disease models. However, translation from the preclinical model into clinical practice has proven to be considerably more difcult. One key challenge being the inability to perform in situ assessment of the hMSCs in continuous culture, where the accumulation of the senescent cells impairs the culture’s viability, diferentiation potential and ultimately leads to reduced therapeutic efcacies. Histochemical β ‑galactosidase staining is the current standard for measuring hMSC senescence, but this method is destructive and not label‑free. In this study, we have investigated alternatives in quantifcation of hMSCs senescence, which included fow cytometry methods that are based on a combination of cell size measurements and fuorescence detection of SA‑β‑galactosidase activity using the fuorogenic substrate, C 12FDG; and autofuorescence methods that measure fuorescence output from endogenous fuorophores including lipopigments. For identifcation of senescent cells in the hMSC batches produced, the non‑destructive and label‑free methods could be a better way forward as they involve minimum manipulations of the cells of interest, increasing the fnal output of the therapeutic‑grade hMSC cultures. In this work, we have grown hMSC cultures over a period of 7 months and compared early and senescent hMSC passages using the advanced fow cytometry and autofuorescence methods, which were benchmarked with the current standard in β‑galactosidase staining. Both the advanced methods demonstrated statistically signifcant values, (r = 0.76, p ≤ 0.001 for the fuorogenic C 12FDG method, and r = 0.72, p ≤ 0.05 for the forward scatter method), and good fold diference ranges (1.120–4.436 for total autofuorescence mean and 1.082–6.362 for lipopigment autofuorescence mean) between early and senescent passage hMSCs. Our autofuroescence imaging and spectra decomposition platform ofers additional beneft in label‑free characterisation of senescent hMSC cells and could be further developed for adoption for future in situ cellular senescence evaluation by the cell manufacturers. Owing to human mesenchymal stromal cells’ (hMSCs) multipotent diferentiation potential, trophic functions and applications in cell and gene therapy1, they have attracted considerable research and clinical interests. In 2006, the International Society for Cellular Terapy (ISCT) proposed a set of minimal criteria to characterize MSCs including cell surface marker expression (must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules), must be plastic-adherent when maintained in standard culture conditions, and must be able to diferentiate into osteoblasts, adipocytes and chondroblasts in vitro2. Based on their high proliferative potential in vitro, hMSCs have been applied exten- sively in cell-based therapy of graf-versus-host disease, liver failure and rejection afer liver transplant, multiple sclerosis and myocardial infarction 3,4. However, variable outcomes of hMSCs transplantation were observed, resulting from difculties in controlling the fate of transplanted cells 5 to reduced therapeutic efcacy afer trans- plantation. Despite the cultures’ compliance to the ISCT defnitions, these variable outcomes can be attributed by the diferent hMSCs tissue sources used6,7, through the diferent expansion procedures and the use of diferent 1Bioprocessing Technology Institute, A*STAR , 20 Biopolis Way, Centros 06-01, Singapore. 2Leeds Institute of Rheumatic and Musculoskeletal Medicine, Leeds, UK. 3Singapore Institute of Manufacturing Technology, A*STAR , 2 Fusionopolis Way, Innovis 08-04, Singapore. *email: [email protected]; [email protected] Scientifc Reports | (2021) 11:1054 | https://doi.org/10.1038/s41598-020-79831-9 1 Vol.:(0123456789) www.nature.com/scientificreports/ media. On top of these factors, replicative senescence 8 of hMSCs also signifcantly impact on the therapeutic efcacy, but is not currently included in MSCs release criteria for their therapeutic use9. Terefore, assessing hMSCs cultures for the presence of senescent cells remains critically important but is not routinely performed in hMSCs manufacturing processes 10. Numerous studies have documented that in hMSCs cultures, the amount of highly proliferative cells declines as the passage number increases, resulting in late passages of large and almost non-proliferative senescent hMSCs11–13. Te study by Wagner et al. on serially passaged hMSCs revealed gradual changes in the global gene and miRNA expression12. Teir study concluded that these senescent-link changes in gene and protein expres- sion were not only associated with senescent passages, but also observed at the start of in vitro expansion. Tus, this accumulation of senescent cells from early passages suggests that identifying senescent cells in early hMSCs cultures can be an important analytical step to ensure the best hMSCs product quality for cell-based therapy. Tough MSCs have been actively applied in industry clinical trials of allogeneic transplantations14, there is still much to understand about MSCs aging and replicative senescence status to improve the long-term safety and efcacy of MSCs engrafment. At the molecular level, retinoblastoma protein (Rb) or p53 pathways triggers the cellular senescence process 15. Additionally, senescent cells typically exhibit senescence-associated β-galatosidase (SA-β-gal) activities, acquire persistent DNA damage nuclear foci (PDDF) that contain DDR proteins ( γ H2AX and 53BP1)16 and secrete growth factors, proteases and cytokines, which some of these biological features can be explained by the senescence-associated secretory phenotype (SASP). Te ISCT criteria released in 20062 is the current standard for regulatory approvals on hMSCs, but factors such as cellular senescence are not included in this standard criteria. Human MSCs attain replicative senescence, a feature likely to occur in industrial-scale MSC expansions, that impair their ability to suppress infammation and reduce their therapeutic efcacy. Other clinical release criteria include microbiology testing and potency assessments10. Among the various methods in characterising senescent hMSCs, the detection of senescence-associated β -galactosidase (SA-β-gal) activity through staining at the optimal lysosomal pH 17,18 is the most contemporary standard. Tis method is closely associated with the accumulation of senescent cells, can be easily applied and detected at near-neutral pH, and was tested as a useful biomarker for detection of senescence in culture and even in vivo in rodents and primates 19. Te SA-β-gal serves as a positive control for other senescence charac- terization methods being developed, and can be more conclusive in senescence characterization in combina- tion with fow cytometry or automated image analysis methods 20. Te detection of higher SA-β-gal activity is associated with an increase in lysosomal mass and accumulation of increased levels of GLB1 mRNA and protein in senescent cells17,21. Cytochemical staining of SA-β-gal is one way to quantify the level of cell senescence as the percentage of senescent cells can be determined through counting the number of blue stained cells in the total population22. However, this method is time consuming and subjective. In contrast to this cytochemical method, a fuorescence-based method (Fig. 1 bottom lef) diferentiates between senescent and non-senescent cell populations more accurately based on incubation with C12 FDG, a fuorogenic substrate for β-galactosidase, as it becomes fuorescent afer entering the cell and cleaved by the enzyme23. Furthermore, the fow cytometric method provides cell size measurement of hMSCs through forward scatter (FSC)24 (Fig. 1 bottom right) and potentially ofers a high-throughput alternative to the cytochemical method to quantitatively evaluate hMSCs senescence. Tis is linked to senescent cells generally displaying fattened and enlarged cell size 1, which can be measured on the FSC channel24. In our previous work, a method based on acquiring and processing native signals from live cells using the label-free technique of autofuorescence spectroscopy has been developed25. Autofuorescence methods have found many applications in biomedical research and diagnosis 26. Its signal comes from a unique class of auto- fuorescent bio-molecules native to cells 27, and these bio-molecules can be diferentiated based on the specifc spectral distribution of their autofuorescence emissions. Of particular interest among these bio-molecules are lipofuscin and lipofuscin-like pigments28, and their correlation with the state of senescence. Lipofuscin is formed by lipids, metals and misfolded proteins, which is especially abundant in nerve cells, cardiac muscle cells and skin29. Lipofuscin-like pigments are distinguished from lipofuscins and are bipartite granules consist- ing of an autofuorescent electron-dense pigment and electron-lucent lipid components30. Both lipofuscins and lipofuscin-like pigment give similar autofuorescence properties and will be measured collectively